The islet of Langerhans plays a key role in glucose homeostasis through regulated hormone secretion. Islet research has focused on the insulin-secreting ?-cells, even though aberrant secretion of another islet hormone, glucagon from ?-cells, exacerbates the pathology of diabetes. Normalization of glucagon action by glucagon receptor antagonism can help diabetic patients maintain euglycemia and avoid hypoglycemic episodes, but progress on this approach has been slowed by numerous side-effects. An alternate approach would be to reduce glucagon secretion, but the mechanism controlling its exocytosis remains controversial. Recent data from our lab and others suggest that the long-standing focus on ?-cell Ca+ signaling may have been misleading, and that regulation of glucagon secretion requires signaling through multiple pathways. This complexity drives an innovative research strategy where we integrate data from mechanistic experiments focused on individual components, while also testing for possible cross-talk between pathways. The loss of glucose-inhibition of glucagon secretion (GIGS) in vivo after ?-cells are destroyed in Type 1 diabetes suggests that interactions between islet cell types are critical to ?-cell function. This has led to models of paracrine signaling where secreted factors from islet ?- and ?-cells constrain ?-cell function. We have shown that insulin and somatostatin work in concert to reduce cAMP and PKA activity, which lowers glucagon secretion, but does not by itself explain GIGS. Preliminary data point to a key role for complexin 2 in linking PKA activity to secretion. We have also shown that a novel juxtacrine pathway, EphA4/7 forward signaling, is activated by ephrinA5 ligands on the ?-cell surface. This leads to F-actin polymerization and decreased glucagon secretion, putatively via RhoA activation. Thus, both paracrine and juxtracrine pathways drive GIGS from islets, but dispersed ?-cells treated with ephrinA5 also exhibit an additional GIGS mechanism, which appears to be intrinsic to the ?-cell. Based on these data, we hypothesize that GIGS requires a synergistic combination of paracrine, juxtacrine, and cell-intrinsic signaling pathways. This hypothesis will be tested via three specific aims: 1) Determine the role of paracrine-mediated PKA-activated phosphorylation of complexin 2 in insulin- and somatostatin- mediated inhibition of glucagon secretion; 2) Determine the role of RhoA activation in the juxtacrine EphA4/7 forward signaling that leads to inhibition of glucagon secretion; 3) Determine the role of EphA4/7 forward signaling in intrinsic ?-cell response to glucose. The multiple intracellular and intercellular signaling mechanisms that we are uncovering will be elucidated by methods that allow precise observation of the pertinent dynamics in living cells and islets. The research plan will also leverage our findings that the mechanisms of GIGS are similar in mouse and human ?-cells, and we will perform parallel experiments across species to the extent possible. These experiments will further our understanding of ?-cell function, which is a critical step towards discovering new potential targets for the regulation of glucagon and treatment of diabetes.